Abstract Alternative splicing has a vital role in generating extensive protein diversity across human cell types. However, misregulation of alternative splicing is commonly found in diseases ranging from developmental disorders to cancer. Despite clear progress in identifying the features, such as cis-acting sequences and trans factors, that predict alternative splicing outcomes, there remains a lack of understanding into key mechanisms of alternative splicing regulation. For instance, there is strong indication that the kinetic rate of splicing is crucial for alternative splicing decisions; however, due to limitations in current approaches for measuring splicing rate, this concept has yet to be directly measured. The overall goal of this proposal is to investigate the variation in splicing kinetics at higher resolution than previously possible and determine how splicing rate is regulated in human cells. I hypothesize that the kinetic rate of splicing is an important regulatory step in determining alternative splicing outcomes. In order to fully test this hypothesis, it is necessary to first expose what regulates splicing kinetics in vivo. By determining the variation and regulation of splicing rates, I will be able to expose how splicing kinetics influence alternative splicing decisions in human cells. First, I will uncover new insights into the variation of co-transcriptional splicing kinetics by probing the splicing rate of 50 constitutive and 50 alternative splicing reactions using a novel approach that measures splicing rates at high resolution. Second, I will reveal how different components of the splicing machinery control splicing kinetics using splicing inhibitors and cell lines with genetic mutations in splicing factors. Third, I will investigate how splicing enhancer and silencer sequences influence the kinetic rate of alternative splicing reactions in order to resolve a connection between splicing kinetics and alternative splicing decisions. Ultimately, the results from this proposal will reveal the variation in co-transcriptional splicing rates in human cells with more accuracy than previously possible and set the foundation for how splicing kinetics are regulated and influence alternative splicing in vivo.